Abstract
A putative zinc-dependent protease (TK0512) in Thermococcus kodakarensis KOD1 shares a conserved motif with archaemetzincins, which are metalloproteases found in archaea, bacteria, and eukarya. Phylogenetic and sequence analyses showed that TK0512 and its homologues in Thermococcaceae represent new members in the archaemetzincins family, which we named AMZ-tk. We further confirmed its proteolytic activity biochemically by overexpression of the recombinant AMZ-tk in Escherichia coli and characterization of the purified enzyme. In the presence of zinc, the purified enzyme degraded casein, while adding EDTA strongly inhibited the enzyme activity. AMZ-tk also exhibited self-cleavage activity that required Zn2+. These results demonstrated that AMZ-tk is a zinc-dependent protease within the archaemetzincin family. The enzyme displayed activity at alkaline pHs ranging from 7.0 to 10.0, with the optimal pH being 8.0. The optimum temperature for the catalytic activity of AMZ-tk was 55°C. Quantitative reverse transcription-PCR revealed that transcription of AMZ-tk was also up-regulated after exposing the cells to 55 and 65°C. Mutant analysis suggested that Zn2+ binding histidine and catalytic glutamate play key roles in proteolysis. AMZ-tk was thermostable on incubation for 4 h at 70°C in the presence of EDTA. AMZ-tk also retained >50% of its original activity in the presence of both laboratory surfactants and commercial laundry detergents. AMZ-tk further showed antibacterial activity against several bacteria. Therefore, AMZ-tk is of considerable interest for many purposes in view of its activity at alkaline pH, detergents, and thermostability.
Highlights
Most organisms typically employ 2–4% of their genetic resources to code for proteases (EC 3.4.1119/21-25/99), which are one of the most abundant classes of enzymes and are involved in a wide range of biological processes, including cell-cycle progression, cell signaling, proliferation and death, protein trafficking and immune response (Deu et al, 2012; Hu et al, 2012)
Thermococcus kodakarensis KOD1, which was kindly donated by the Japan Collection of Microorganisms, RIKEN BioResource Center, Japan, was used to isolate genomic DNA, and it was cultured in 280 Thermococcus medium (Fukui et al, 2005)
Following the proteins in the Thermococcaceae family, AMZ-tk is similar to archaemetzincins in other organisms, such as the two archaemetzincins in humans and TK1178 in T. kodakarensis KOD1, which was annotated as archaemetzincin
Summary
Most organisms typically employ 2–4% of their genetic resources to code for proteases (EC 3.4.1119/21-25/99), which are one of the most abundant classes of enzymes and are involved in a wide range of biological processes, including cell-cycle progression, cell signaling, proliferation and death, protein trafficking and immune response (Deu et al, 2012; Hu et al, 2012). Proteases differ in properties such as substrate specificity, active site and catalytic mechanism. Metalloproteases contain a metal ion at their active site that acts as a catalyst in the hydrolysis of peptide bonds. Most metalloproteases require zinc (Zn2+), but other transition metals have been found at active sites, such as cobalt (Co2+) and manganese (Mn2+) (Holmquist and Vallee, 1974; Häse and Finkelstein, 1993). Archaemetzincin or archaelysin (MEROPS family M54.001) is a zinc-dependent aminopeptidase that contains the consensus zinc-binding sequence HExxHxxGx3Cx4CxMx17CxxC and a conserved Met residue at the active site. As more genome sequences have become available, archaemetzincins have been identified in plants; for example, Pyrus bretschneideri (XP_009350639.1), which was annotated as an uncharacterized protein (Wu et al, 2013)
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